Role of Protein-Lipid Bilayer Interactions in alpha-Synuclein Aggregation This proposal is in response to the NIA's Pilot Research Grant Program on Research Objective #7--Protein Modification, Aggregation and Degradation in Aging and Age-related Diseases. Parkinson's Disease (PD) is an age-related, neurodegenerative disease characterized by resting tremor, muscular rigidity, postural instability, and bradykinesia. Although the molecular mechanism responsible for PD is currently unknown, the soluble neural protein, alpha-synuclein (aS), is believed to be involved in the pathological cascade, alphaS is the major constituent of Lewy bodies, intracellular fibrillar aggregates that are a pathological hallmark of PD. AlphaS is known to self-aggregate in vitro into multimers and fibrils that resemble those found in Lewy bodies. Numerous in vivo and in vitro studies have also demonstrated that alphaS co-exists with lipids in Lewy bodies, and that aS directly interacts with phospholipids in vesicle or cell membranes. At the present time, there are two proposed mechanisms for aS aggregation and fibril formation in neural cells. The first mechanisms is that soluble aS protein adheres to the cell surface via lipid-protein interactions, and serves a seed or nucleus for aggregation and fibril formation. The second mechanism is that the natural state of alphaS is to be associated or bound to lipids such that the protein is not free to aggregate with other soluble proteins. The objective of this research is to distinguish between the two proposed mechanisms. Our hypothesis is that the interactions between alphaS and lipids play a role in protein aggregation. The atomic force microscope (AFM) will be used in this study to characterize the initial interactions between alphaS and supported lipid bilayers, and to determine how these interactions affect protein aggregation. The objective of this research will be achieved through the following specific aims: 1) Characterize the strength of the interactions between alphaS and supported lipid bilayers containing various mixtures of anionic and neutral phospholipids representative of neural cell membranes. 2) Determine if electrostatic interactions mediate protein-lipid binding. 3) Evaluate aS aggregation on lipid bilayers as a function of phospholipid composition. A correlation between the strength of protein-lipid interactions and protein aggregation will directly support our hypothesis of lipid-induced self-aggregation of alphaS. The AFM, which can provide unique quantitative information on the magnitude of biological interactions, will be used in this study to evaluate protein-lipid bilayer interactions. Protein will be immobilized directly on the tips of AFM cantilevers and used to probe supported lipid bilayers in buffer solutions. The AFM will also be used to observe the formation of alphaS aggregates on lipid bilayers. Ultimately, results from this study will be used to develop therapies that prevent alphaS aggregation.